Scrub typhus is a potentially fatal disease that is endemic in the Asia-Pacific region, where 1 billion people are at risk for infection and 1 million new cases are reported annually. Scrub typhus is treatable with antibiotics, but reinfections are common and decreased efficacy of antibiotics has been increasingly reported. A recent outbreak among U.S. Marines training at a military base in Japan, and the prevalence of scrub typhus in Afghanistan and Pakistan where U.S. troops are deployed, echoes the risk of U.S. soldiers for the disease. The etiologic agent is the trombiculid mite-transmitted obligate intracellular bacterium, Orientia tsutsugamushi. Despite the global health threat that it poses, O. tsutsugamushi is severely understudied. Indeed, how this pathogen manipulates eukaryotic host cell functions to facilitate its intracellular survival is poorly understood. An emerging theme among many intracellular bacterial pathogens is that they translocate ankryin repeat-containing proteins (Anks) into eukaryotic host cells. The ankryin repeat is one of the most common protein-protein interaction motifs in nature. Anks of other bacterial and viral pathogens traffic t distinct subcellular locations where they interact with host target proteins and mimic or interfere with host cell functions to facilitate pathogen survival. The O. tsutsugamushi genome carries 38 ank genes, and we have determined that all 38 are expressed during infection of mammalian host cells in vitro. Genes encoding Ank4, Ank9, and Ank12_1 are induced at 1 h post-infection, Ank6 at 4 h, and Ank13 at 8 h. We hypothesize that Ank4, Ank6, Ank9, Ank12_1, and Ank13 are important for O. tsutsugamushi to establish infection and have prioritized these proteins for functional studies. Genes encoding ank1, ank5, and ank17 are also expressed during infection and carry coiled-coil domains. The coiled-coil domain is a signature protein-protein interaction motif of numerous bacterial pathogen effectors that mediates interaction with host cell targets. Because Ank1, Ank5, and Ank17 carry both ankyrin repeat and coiled-coil motifs, we will include them in our functional studies. In Aim 1, we will assess if the eight Anks of interest perform distinct effector functions by ectopically expressing the proteins in mammalian host cells and identifying the subcellular locations to which they traffic. To determine if they interact with hos cell factors that are critical for O. tsutsugamushi intracellular survival, we will assess whether ectopically expressed Anks competitively inhibit Orientia survival. In Aim 2, we will capture and identify host cell target proteins that interact with Orientia Anks using in vivo coprecipitation ad affinity chromatography. The proposed work will fill a considerable knowledge gap of how an understudied pathogen of global biomedical importance facilitates its intracellular survival. Since antibiotic resistance among O. tsutsugamushi isolates is increasing and reinfections are common, defining the functional roles of Anks will potentially aid the design of small molecule inhibitors that target specific Anks to treat scrub typhus.